Unassigned or nonsense codons in Micrococcus luteus.

We previously reported that in Micrococcus luteus, a Gram-positive eubacterium with high genomic G + C content, certain codons ending with A did not appear in coding frames, including termination sites, and tRNAs that translate these codons were not detected. These facts suggest that at least some of them are unassigned (nonsense) codons, i.e. not assigned to any amino acid or to any stop signal. We have investigated whether AGA and AUA, universal Arg and Ile codons, respectively, are really unassigned codons by using a cell-free extract prepared from M. luteus and synthetic messenger RNAs. Translation of synthetic mRNA containing in-frame AGA codons does not result in "read-through" to codons beyond the AGA codons, i.e. translation ceases at codon AGA. Essentially the same result was obtained with mRNA containing AUA in-frame. A sucrose-gradient centrifugation profile of the reaction mixture has shown that practically all of the peptides that have been synthesized are attached to 70 S ribosomes. When in-frame AGA or AUA codons are replaced by UGA codons in mRNA, no read-through occurs beyond UGA, just as in the case of AGA or AUA. However, the synthesized peptide is released from the 70 S ribosomes. These data suggest that AGA and AUA are unassigned codons and differ from UGA in that they are not used for termination.